PERMEABILITY OF THE CONJUNCTIVA AND THE SCLERA

IV. PERMEABILITY OF THE CONJUNCTIVA AND THE SCLERA

The recognition that noncorneal penetration may be a productive route of intraocular entry for some drugs has triggered extensive research to under- stand the barrier properties of the conjunctiva and the sclera and the physicochemical determinants of drug permeability across these membranes. In vitro and in situ permeability studies have proven to be a useful approach in estimating drug transport for in vivo conditions. In general, permeability characteristics of ocular membranes correlate well with the intraocular absorption of drugs.

A. Methodology Four methods have been commonly used for measuring permeability across

ocular membranes. The first method is based on the measurement of steady- state permeability of drugs across the isolated ocular membrane using a side-by-side diffusion cell or a modified two-chamber Ussing apparatus.

The Noncorneal Route in Ocular Drug Delivery 345 Accordingly, the membrane permeability coefficients may be calcu-

lated using the following equation: P¼V R

the slope of the linear part of the amount of drug accumulated versus time curve, V R is the volume of the receiver chamber, A is the surface area of the

mounted membrane, and C D is the initial concentration of the drug in the donor chamber. This method assumes sink conditions. As pointed out by Maurice (32) in his critique of such techniques, it is important to ensure that the in vivo characteristics of the membranes are maintained as closely as possible. This requires care during the dissection to avoid folding or damage to the surfaces of the membranes and preservation of the tissue during the course of the experiment using physiologically relevant buffer systems (e.g., oxygenated glutathione bicarbonate Ringers solution) to maintain tissue viability. It is also important to measure, confirm, and report the integrity of the barrier based on electrical properties, hydration level, and diffusion of marker substances. Another alert is interspecies differences and caution in overinterpreting data obtained from animals to humans.

The second method is in situ perfusion technique that enables quanti- tation of drug uptake in live animals. This method was originally utilized for measuring corneal permeation and uptake of drugs (110,111) and subse- quently applied to study the conjunctival and scleral uptake of various compounds (10,11,16). The technique involves affixing a cylindrical well on the surface of the eye near the corneoscleral junction using surgical adhesives (Fig. 4). A drug solution is then placed either inside the well bathing the cornea or outside the well bathing the remainder of the con- junctival sac. The rate of mass flux in the system can be described by:

dC s V s = dt ¼ Cl s : C s where C s is the concentration of drug in the systeml, V s is the volume of fluid

in the reservoir and Cl s is a clearance parameter describing the loss of drug from the system. This method can be utilized to study the effect of physical- chemical drug properties while avoiding some of the problems associated with in vitro studies on isolated membranes as previously described. The primary disadvantage is the difficulty in extracting a true permeability coef- ficient or mechanistic information from such experiments.

A third is a flow-through permeation chamber where the excised tissue is mounted horizontally method (43,46,112). A small volume of the drug solution is applied on the external surface of the membrane and the internal surface is perfused with a physiological receptor solution. In this method the flux across the membrane can be calculated using the following equation:

346 Ahmed

Figure 4 In situ corneal blocking technique.

where V is the volume of the receiver compartment, C is the concentration in the receiver compartment, S is the exposed surface area, J is the flux at the membrane surface on the receptor side, Q is the flow rate out of the receptor compartment, and t is the time. This method is appropriate for the measurement and prediction of transient transport across the ocular mem- brane simulating more realistic ocular drug delivery scenarios. For example, it has been shown that the predictions of transscleral transport based on steady-state measurements may significantly overpredict the amount of drug delivered into the eye because the lag time for transport across the sclera is similar or longer than the drug-sclera contact time from an eye drop (43). Drug binding to the sclera may also prolong the lag time. A disadvantage of non–steady-state experiments is that the data treatment and mathematics is complicated.

The fourth method is the use of cell culture and physical models. During the past decade advances in cell culture techniques have resulted in the development of a primary culture model of rabbit conjunctival epithe-

The Noncorneal Route in Ocular Drug Delivery 347 lial cells exhibiting tight barrier properties (112–115). There has been an

attempt to develop physical models to describe the transport of molecules through the cornea and the sclera by taking into account the ultrastructure of these tissues (44). The use of intestinal tissues to predict ocular perme- ability and ex vivo models based on isolated perfused tissue has also been reported (118). These techniques may offer an alternative to the use of animals in research and an opportunity for gaining a greater mechanistic understanding of transport processes. However, the practical application and scope of these methods remain to be determined.

B. Permeability of the Sclera and Conjunctiva

1. Sclera The permeability of the sclera to small molecules is comparable to that of

the corneal stroma. There was no significant difference between the corneal stroma and the sclera of the beef eye in their permeability to a variety of small solute (24). Even large molecules can move across the sclera by the way of perivascular spaces but also by diffusion through interfibrillary spaces (38). Maurice and Polgar measured the movement of a number of anionic and cationic dyes, as well as small ions, proteins, and biologically active molecules across the isolated beef sclera using a two-chamber diffu- sion cell (39). Only anionic dyes of small molecular weights were able to cross the sclera, but the sclera offered little obstacle to the penetration of drugs into the eye.

Prausnitz and Noonan (24) recently published a comprehensive data- base of ocular tissue permeability measurements found in a review of the literature. There was no apparent dependence on distribution coefficient but

a strong dependence of penetrant size on scleral permeability. The scleral permeability to tracers of various molecular sizes in rabbit and humans is shown in Table 3. Ambati et al. (47) showed that molecular radius was a better predictor of scleral permeability than molecular weight, and large molecules, such as IgG, diffuse across the sclera in a manner consistent with porous diffusion through a fiber matrix. Edwards and Prausnitz sup- ported this assertion with a fiber matrix model to predict the permeability of the sclera to water and solute (44). Unlu and Robinson (45) observed that the scleral permeability of radiolabeled hydrocortisone and mannitol across the isolated rabbit sclera was five times greater than the corneal permeabil- ity. The low activation energy of scleral transport of hydrocortisone sug- gested an aqueous pore pathway. Hamalainen (30) used a modified two- chamber Ussing apparatus to characterize quantitatively the paracellular permeation routes in the rabbit cornea, conjunctiva, and sclera using poly-

348 Ahmed Table 3 Permeability of Sclera to Tracers of Various Molecular Sizes in

Rabbits and Humans Molecular

Permeability coefficient Tracer

Molecular

weight (D)

radius (nm)

cm/s (SD) Rabbit

Human Water

18 54.4 919) a 44.6 (13) a Mannitol

28.3 (3.7) d Sucrose

42.2 (13.7) e 21.6 (6.0) b Hydrocortisone

21.8 (4.3) d Dexamethasone

12.7 (2.3) a 18.2 (5.8) a 23.5 (7.7) b

0.5 84.5 (16.1) c Carboxyfluorescein

Sodium fluorescein

13.0 (3.4) a 11.8 (1.37) a Dextran, 4 kDa

1.3 25.2 (5.1) c Inulin

5000–5,250 2.54 (0.35) e 9.0 (2.2) b Dextran, 10 kDa

6.4 (1.7) b Dextran, 20 kDa

3.2 6.79 (4.18) c Dextran, 40 kDa

4.5 2.79 (1.58) c 4.9 (2.4) b Dextran, 70 kDa

6.4 1.39 (0.88) c 1.9 (0.4) b Dextran, 150 kDa

8.25 1.34 (0.88) c SD = Standard deviation.

a From Ref. 46. b From Ref. 42.

c From Ref. 47. d From Ref. 45.

e From Ref. 40.

ethylene glycol (PEG) oligomers. The scleral permeability was 15–25 times more than that of the cornea but about half that of the conjunctiva. The permeability of PEGs decreased linearly with increasing molecular weight,

cm/s for PEG cm/s for PEG 942. There have been several reports comparing the permeability of the sclera, conjunctiva, and cornea to penetrant lipophilicity. The comparative permeability of a series of b-blockers across isolated rabbit ocular mem- branes is shown in Table 4. Ahmed et al. (40) showed that the conjunctival and corneal permeabilities for timolol were comparable but fourfold lower than the scleral permeability. Sasaki and coworkers (54) reported that the in vitro permeability of a series of b-blockers across the isolated rabbit sclera and conjunctiva was not affected by penetrant lipophilicity. Edelhauser and Maren (41) compared the scleral versus corneal permeability of the carbonic

The Noncorneal Route in Ocular Drug Delivery 349 Table 4 Differential Permeability of b-Blockers Across Isolated Ocular

Membranes in Rabbit 6 )

Compound MW

Conjunctiva Sclera Atenolol

71 Source : Adapted from Ref. 24.

anhydrase inhibitors ethoxzolamide and methazolamide and concluded that the permeability was six times greater in the sclera than in the cornea for the less lipophilic methazolamide but similar for the more lipophilic ethoxzola- mide.

2. Conjunctiva Unlike the sclera, the conjunctiva shares an important attribute with the

cornea in that both contain an outer lining of stratified squamous epithelium continuous with each other at the corneoscleral limbus. However, the cornea is avascular while the conjunctival epithelium overlies a loose, highly vas- cular connective tissue, the substantia propria.

Characteristic of any epithelial tissue, both paracellular and transcel- lular transport is possible across the conjunctiva. In both the cornea and the conjunctiva, lipophilic drugs prefer the transcellular route, while the para- cellular route is preferred by hydrophilic drugs (36). The ratio of the perme- ability coefficients of a series of b-adrenergic blockers in the cornea and those in the conjunctiva exhibited a sigmoidal correlation with log partition

350 Ahmed coefficient, as shown in Figure 5 (51). This indicates that while the conjunc-

tiva is leakier than the cornea, the differential permeability of the conjunc- tiva to the cornea is higher for hydrophilic compounds than for lipophilic compounds. The data presented in Table 5, which shows that the perme- ability of peptides in conjunctiva is higher than in the cornea, support this conclusion (52,53).

Several authors have studied the effect of molecular size on conjuncti- val permeability. Horibe et al. (31) characterized the conjunctival perme- ability to polar solutes ranging from 182 to 167,000 daltons in molecular weight and concluded that solutes up to 40 kDa traverse the conjunctival epithelial barrier primarily by restricted diffusion through equivalent pores of 5.5 nm. Polar solutes of greater than 70 kDa may cross the barrier primarily via nondiffusional pathways, such as nonspecific endocytosis. Huang et al. (27) and Kahn et al. (28) also studied the solute size effect on conjunctival penetration. They estimated the limiting size of solutes that can pass the conjunctival barrier by paracellular transport at between 20 and 40 kDa. Hamalainen et al. (29) reported that the conjunctival epithelia in the rabbit have 2 times larger pores and 16 times higher pore density than

10 mm 2 mm 2 in rabbit conjunctiva. Finally, there is some evidence that human conjunctiva may be more permeable to hydro- philic solutes than rabbit conjunctiva (32)

Figure 5 Permeability coefficient ratio (cornea/conjunctiva) as a function of the log partition coefficient

The Noncorneal Route in Ocular Drug Delivery 351 Table 5 Permeability Ratio of Peptides Across Ocular Membranes in Rabbit Permeability ratio

Conjunctiva/Cornea Diglycine

Compound MS

1182 47 Source : Adapted from Refs. 36, 53.